PAKs are required for the degradation of Vac17 and the release of the vacuole from Myo2. (A) Vac17 levels are elevated in dma1Δ and cla4^ts ste20Δ mutants. The cla4^ts ste20Δ mutant was grown at either 24°C or shifted to 37°C for 3 h before lysis. Pgk1 was used as a loading control. Molecular mass is shown in kilodaltons. (B–E) Loss of PAK function results in mislocalization of the vacuole (FM4-64; B and D) and accumulation of Vac17-GFP (B) at the bud tip (arrowheads) or mother-bud neck (arrows). Wild-type (WT) andcla4^ts ste20Δ cells were transformed with Vac17-GFP (B) or Myo2-Venus (D). After FM4-64 labeling, cells were chased either at 24°C for 3 h or 24°C for 90 min and then 37°C for 90 min before imaging. DIC, differential interference contrast. (C and E) Quantification of >35 large-budded cells per condition per n. Error bars indicate SEM. n = 3. *, P < 0.05; **, P < 0.01; two-tailed Student’s t test.